Traditional therapies, including surgery, radiation, and chemotherapy, have played a significant role in the treatment of breast cancer. However, it is unlikely that these modalities alone can be further optimized to cure the 44,000 women/year who currently fail these modalities. Immunotherapy is another modality that has recently shown promise for the treatment of breast cancer. In particular, the herceptin antibody that is specific for the HER-2/neu (neu) protein results in cancer repression in patients who are resistant to the other treatment modalities. Vaccines, a form of immunotherapy, have the advantage over other treatments in they are specific can amplify the response to meet the burden of existing cancer, and can induce immune memory for the cancer if it recurs. In addition, vaccines have the potential to induce immunity capable of preventing cancer development A few vaccines have already resulted in cancer remissions for patients with small disease burdens. However, to date, the majority of vaccines have been developed using tumor cell lines that are transplanted into non-tolerized mice, a situation that is very different from the tolerance that occurs to naturally arising human tumors. In this proposal, we will test the hypothesis that combining immune modulating antibodies that alter the natural signaling patterns of key T cell regulatory molecules together with antigen-specific vaccination can enhance the potency of neu-targeted vaccines and result in improved treatment and prevention strategies. We will use the neu transgenic mice that naturally over-express the neu protein and spontaneously develop mammary tumors. We have characterized these mice and found that they demonstrate neu-specific peripheral tolerance similar to what is observe in patients with breast cancer. Using this clinically relevant model, we have generated preliminary data demonstrating that neo-specific vaccine approaches an induces immunity potent enough to overcome peripheral tolerance and delay tumor development. In this proposal, we will: 1) Modify and optimize the most potent neu targeted vaccine; 2) Evaluate antagonist antibodies against CTLA-4, and agonist antibodies against CD40 and OX-40, three molecules that are critical for T cell activation and down-regulation, for their ability to enhance systemic anti-tumor immune responses; 3) Evaluate optimized vaccine plus immune modulating approaches that demonstrate improved anti-tumor activity in the less stringent transplantable tumor model for the ability to prevent spontaneous tumor development; 4) Evaluate optimized vaccine plus immune modulating approaches for the ability to overcome neu-specific tolerance under circumstances of large naturally developing tumor burdens. Although neu is the immune target in these studies, it services a model antigen for testing vaccine treatment and prevention strategies. The final goal of this proposal is to use information gained from these studies to design clinical trials aimed at both the treatment and prevention of breast cancer. The success of these studies may define a new paradigm for cancer prevention.